Significance

G protein-coupled receptors (GPCRs) form a family of signaling molecules in the membrane of cells that plays a key role in transduction of cellular responses. Little is known about how rapidly GPCRs can be activated. While the “light receptor” rhodopsin in the eye activates within 1 ms, other GPCRs are thought to activate much slower. We use two entirely different techniques with advanced time resolution to activate a dimeric metabotropic glutamate GPCR: UV light-triggered uncaging of ligand in intact cells and piezo-driven ligand application in outside-out patches. We demonstrate initial conformational rearrangements within ≈1 ms that are followed by much slower (≈20 ms) activation in the transmembrane domain. Thus, the initial activation of a nonvisual GPCR proceeds with millisecond speed.

Abstract

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the “light receptor” rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ∼1 ms, while all other receptors are thought to become activated within ∼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 ∼ 1–2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 ∼ 20 ms). Receptor deactivation occurred with time constants of ∼40 and ∼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including—in addition to the inactive and active states—at least two metastable intermediate states.

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